中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (7): 1103-1108.doi: 10.3969/j.issn.2095-4344.1059

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

载脂蛋白E基因敲除(APOE-/-)小鼠基因的鉴定方法及模型应用

张文将1，2，易  健3，贾  平1，陈博威1，王  勇2，刘柏炎1   

  1. (1湖南中医药大学，湖南省长沙市  410208；2益阳医学高等专科学校，湖南省益阳市  413000；3湖南中医药大学第一附属医院，湖南省长沙市  410007)
  • 收稿日期:2018-10-07 出版日期:2019-03-08 发布日期:2019-03-08
  • 通讯作者: 刘柏炎，博士，教授，博士生导师。湖南中医药大学，湖南省长沙市 410208
  • 作者简介:张文将，1986年生，男，湖南中医药大学2016级在读博士。
  • 基金资助:

    湖南省科技计划项目(2018JJ2412)，项目负责人：张文将；湖南省中医药科研计划项目(201870)，项目负责人：张文将；湖南省教育厅资助科研项目(17B271)，项目负责人：张文将

Genotype identification and model application of apolipoprotein E knockout mice

Zhang Wenjiang1,2, Yi Jian3, Jia Ping1, Chen Bowei1, Wang Yong2, Liu Baiyan1   

  1.  (1Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China; 2Yiyang Medical College, Yiyang 413000, Hunan Province, China; 3the First Affiliated Hospital of Hunan University of Chinese Medicine, Changsha 410007, Hunan Province, China)
  • Received:2018-10-07 Online:2019-03-08 Published:2019-03-08
  • Contact: Liu Baiyan, MD, Professor, Doctoral supervisor, Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China
  • About author:Zhang Wenjiang, Doctoral candidate, Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China; Yiyang Medical College, Yiyang 413000, Hunan Province, China
  • Supported by:

    the Science and Technology Project of Hunan Province, No. 2018JJ2412 (to ZWJ); the Science Research Program of Hunan University of Chinese Medicine, No. 201870 (to ZWJ); the Project of Education Department of Hunan Province, No. 17B271 (to ZWJ)

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摘要：

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文题释义：
纯合子：是指同一位点上的2个等位基因相同的基因型个体，即遗传因子组成相同，如AA，aa。
野生型基因组：目前的研究把从大自然中获得的个体，也就是非人工诱变的个体，作为野生型，那么它所携带的就是野生型基因组。
摘要
背景：载脂蛋白E基因敲除小鼠是广泛应用于动脉粥样硬化研究的理想模型，也有用于其他疾病的相关报道，但是缺乏对其模型应用的总体概括。
目的：系统总结载脂蛋白E基因敲除小鼠在疾病模型中的应用情况。
方法：将南京大学生物模式中心所引进的SPF级载脂蛋白E基因敲除小鼠雌雄各6只饲养于SPF实验室，按照1∶1搭配进行保种繁殖，采用煮沸裂解法以提取鼠尾组织基因组DNA，采用PCR法检测其基因型。将基因鉴定为载脂蛋白E基因敲除雄性小鼠10只普通饲料喂养至8周龄，继而予以西方饮食饲料(北京华阜康公司提供，型号：H10141，脂肪21%，胆固醇0.15%)连续喂养至25周龄，同时选用8周龄C57/6 J野生型雄性小鼠10只普通饲料喂养至25周龄。
结果与结论：①基因鉴定：琼脂糖凝胶电泳显示载脂蛋白E基因敲除小鼠PCR产物与预期的目的基因片段的大小一致；②相关指标变化：与野生小鼠相比，载脂蛋白E基因敲除小鼠的体质量、肝脏质量、脾脏质量、腹部脂肪质量、肝指数、血清中总胆固醇、三酰甘油、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、谷丙转氨酶及谷草转氨酶明显升高(P < 0.05)；③组织学改变：载脂蛋白E基因敲除小鼠苏木精-伊红染色主动脉弓、主动脉瓣、颈总动脉部位有明显的斑块，野生鼠无斑块；载脂蛋白E基因敲除小鼠肝脏苏木精-伊红染色有大小不等的空泡，细胞核被挤到一边，野生鼠肝脏核大居中，胞浆红染，无空泡现象；④结果证实，实验采用煮沸裂解法以提取DNA，采用PCR法检测成功鉴定了载脂蛋白E基因敲除小鼠的基因型。载脂蛋白E基因敲除小鼠是研究动脉粥样硬化、脂肪肝、高血脂、高胆固醇血症及肥胖等许多疾病的理想模型。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程
ORCID: 0000-0002-8990-4166(张文将)

关键词: 载脂蛋白E基因敲除, 动脉粥样硬化, 基因敲除, 鉴定, 脂肪肝, 高血脂, 肥胖, 阿尔茨海默病, 模型, 小鼠, 组织工程

Abstract:

BACKGROUND: Apolipoprotein E knockout mice are an ideal model of atherosclerosis, which has been reported in the other diseases.
OBJECTIVE: To systematically summarize the application of apolipoprotein E knockout mice in disease models.
METHODS: Six male and six female APOE-/- mice introduced from the Biomedical Research Center of Nanjing University were bred in each cage at 1:1 in the SPF laboratory. The genomic DNA was extracted from mouse tail tissue by the method of boiling lysis, and the genotypes were detected by PCR. The APOE-/- mice were fed with normal diet for 8 weeks, and then fed with a Western diet (provided by Beijing Huafukang Bioscience Co., Ltd., No. H10141, 21% of fat, 0.15% of cholesterol) to 25 week old. At the same time, 10 8-week-old C57/6J mice were fed with a normal diet to 25 weeks old.
RESULTS AND CONCLUSION: Genetic identification: agarose gel electrophoresis results indicated that the size of molecular weight of the PCR products from APOE-/- gene knockout mice was consistent with the expected target gene fragment and of different genotypes successfully. Changes of related indicators: compared with the wild type mice, the body mass, liver weight, spleen weight, abdominal fat weight, liver index, and serum levels of total cholesterol, triacylglycerol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, alanine transaminase, and aspartate transaminase in the APOE-/- mice were significantly increased (P < 0.05). The histological changes: APOE-/- mice showed obvious plaques in the aortic arch, aortic valve and common carotid artery by hematoxylin-eosin staining, but no plaques in the wild type mice. The liver of APOE-/- mice had vacuoles of varying sizes, and the nucleus was pushed aside. The nucleus of wild mice liver was in the middle, the cytoplasm was red, and there was no vacuole phenomenon. These results suggest that PCR can fast and reliably identify the genotypes of apolipoprotein E knockout mice using genomic DNA extracted by boiling lysis. APOE-/- mice are ideal models for studying atherosclerosis, fatty liver, hyperlipidemia, hypercholesterolemia, obesity and many other diseases.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Tissue Engineering, Atherosclerosis, Fatty Liver, Hyperlipidemias

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